Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.
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PMID:The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein. 1 96

The binding of Ca2+ to a previously described phosphoprotein from human parotid saliva, protein A [Bennick (1975) Biochem J. 145, 557-567] was studied by means of equilibrium dialysis. In 5 mM-Tris/HC1 buffer, pH7.5, protein A bound 664nmol of Ca/mg of protein. Km was determined to be 181 muM and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ apparently occurs to side-chain carboxyl groups in the protein, but protein phosphate is of minor if any importance in calcium binding. Hydrolysis of protein A by trypsin and collagenase or heating of the protein at 60 degrees or 100 degrees C did not affect Ca2+ binding. The Ca2+ binding decreases with increased concentration of the dialysis buffer and on the addition of SrCl2, or MgCl2 or MnCl2 to the dialysis buffer. Protein A does not aggregate in the presence of Ca2+, since the s20,w was identical when determined in the presence (1.30S) and absence (1.35S) of CaCl2. By use of a specific antiserum to protein A it was found that protein C [Bennick & Connell (1971) Biochem. J. 123, 455-464] and perhaps minor related components cross-reacted with protein A. No other salivary proteins showed immunological similarity. Proteins A and C were also present in submandibular saliva. The possible functions of protein A are discussed.
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PMID:The binding of calcium to a salivary phosphoprotein, protein A, common to human parotid and submandibular secretions. 18 Sep 80

Protein C is a vitamin K dependent protein present in bovine plasma (Stenflo, J. (1976), J. Biol. Chem. 251, 355). It is a glycoprotein (mol wt approximately 62 000) composed of a heavy chain (mol wt 41 000) and a light chain (mol wt 21 000). The heavy chain has an amino-terminal sequence of Asp-Thr-Asn-Gln and contains nearly three-fourths of the carbohydrate. The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe. Incubation of protein C with either factor X activator from Russell's viper venom or trypsin resulted in the cleavage of an Arg-Ile bond between residues 14 and 15 of the heavy chain. Concomitant with this cleavage was the formation of a serine enzyme which was inhibited by diisopropyl phosphorofluoridate. Liberation of the tetradecapeptide decreased the molecular weight of the heavy chain from about 41 000 to 39 000 and resulted in the formation of a new amino-terminal sequence of Ile-Val-Asp-Gly in the heavy chain. No change in the molecular weight of the light chain was observed during the activation reaction. These results indicate that protein C, like the four vitamin K dependent coagulation proteins, exists in plasma in a precursor form and is converted to a serine protease by hydrolysis of a specific Arg-Ile peptide bond. The biological substrate for the enzymatic form of protein C and the physiological mechanism whereby protein C is converted to a serine enzyme are not known.
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PMID:Proteolytic activation of protein C from bovine plasma. 99 Feb 50

Plasma from women taking combined oral contraceptives and cold-activated plasma contain proteases which cleave chromogenic substrates in protein C assays in the absence of protein C activators such as Protac. This spontaneous activity makes a background substraction necessary and makes protein C (PC) assays less accurate. We investigated two commonly used substrates < Glu-Pro-Arg-pNA (S-2366) and 2AcOH.H-D-Lys(Cbo)-Pro-Arg-pNA (PC substrate) and found that cold-activated normal and protein C-deficient plasmas gave absorbance values up to 300 times higher than buffer blanks. FXIa cleaves these substrates but activity was not blocked by corn or lima bean trypsin inhibitors, soy bean trypsin inhibitor (SBTI), hirudin or epsilon-amino-n-caproic acid (EACA). Kaolin activation of normal, FXI, FIX, FVIII, FVII and protein C-deficient, but not of FXII or prekallikrein (PKK)-deficient plasmas led to cleavage of chromogenic substrate for protein C. The protein C substrates were cleaved by purified kallikrein and alpha- and beta-FXIIa. Immunoabsorption with alpha 2-macroglobulin (alpha 2M) antibodies removed 60% of the alpha 2M and 70% of the activity on PC Substrate. Gel filtration of normal plasma on Sephadex G-150 gave a single peak of protein C activity and antigen in the included volume. After cold activation of the fractions, a second protein C-like peak appeared in the void volume, but with no detectable protein C antigen. This peak coincided with alpha 2M (chromogenic and ELISA) and plasma kallikrein (S-2302), but FXII (measured with a substrate insensitive to kallikrein) eluted separately.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contact factor proteases and the complexes formed with alpha 2-macroglobulin can interfere in protein C assays by cleaving amidolytic substrates. 128 Apr 70

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.
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PMID:Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin. 132 50

A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary binding and/or protecting domain on other portions of the vWF subunit (potentially destroyed during the proteolysis of vWF) is highly unlikely.
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PMID:Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely. 153 49

Widespread intravascular coagulation is common in patients with sepsis. Coagulation abnormalities may result from exposure to endotoxin, from tumor necrosis factor alpha or interleukin 1 release, or from the actions of a more specific mediator, such as vascular permeability factor. The result is marked activation of the contact and coagulation systems; simultaneously, there is decreased fibrinolysis and depressed levels of the inhibitors of the contact and coagulation systems. Multiple agents are being studied to correct these abnormalities. Antithrombin III holds promise because it inhibits a number of factors important in contact and coagulation activation, not just thrombin. Plasminogen activators may prove helpful in increasing fibrinolysis during sepsis; because they have been associated with rebound thrombin generation, however, plasminogen activators may be most effective if used in conjunction with hirudin or a synthetic hirudin analogue. Bradykinin may offset hypotension in sepsis. Protein C may inhibit thrombin formation and also complex with plasminogen activator inhibitor 1, thereby promoting fibrinolysis. Other agents that may prove effective include alpha 1-antitrypsin Pittsburgh, C1-esterase inhibitor, monoclonal antibodies to contact factors, soybean trypsin inhibitors, thrombomodulin, prostaglandin I2, and aprotinin. There are no data to support the use of heparin or fibronectin, except in limited circumstances.
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PMID:Modulators of coagulation. A critical appraisal of their role in sepsis. 162 18

In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192----Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192----Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.
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PMID:Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. 167 22

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.
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PMID:A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation. 173 24

Smooth muscle caldesmon was phosphorylated by protein kinase C up to 1.90 mol P/mol caldesmon. Phosphorylated caldesmon was completely digested by trypsin and the produced phosphopeptides were purified by C-8 and C-18 reverse phase chromatography. Four phosphopeptides were determined and two phosphoserines were identified. Both were localized in the C-terminal domain at serine-587 and serine-726. By following the time course of phosphorylation, serine-587 was found to be the preferred site. Effects of the phosphorylation of caldesmon by protein C on the inhibition of acto-H-meromyosin ATPase activity was also examined. While unphosphorylated caldesmon inhibited the ATPase activity by 60%, phosphorylated caldesmon hardly inhibited the ATPase activity. Therefore, it was concluded that the phosphorylation at serine-726 and serine-587 reverses the inhibitory activity of caldesmon.
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PMID:Determination of the phosphorylation sites of smooth muscle caldesmon by protein kinase C. 189 46


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